3654
BIOCHEMISTRY
SWENSON AND RITCHIE
Enthalpy of Nucleotides Binding to Myosin? C. A. Swenson* and P. A. Ritchie
ABSTRACT:
The enthalpies of binding adenosine 5’-diphosphate (ADP) and 5’-adenylyl imidodiphosphate [AMP-P(NH)P] to rabbit skeletal myosin have been measured in Pipes and Tris buffers at pH 7.8 and 15 O C . For ADP the enthalpy of binding was exothermic, whereas the enthalpy of binding AMPP(NH)P, a nonhydrolyzable ATP analogue, was small and
endothermic. For the reaction of ATP and myosin, the development of enthalpy was resolved into two phases: a fast endothermic phase, which is the summation of binding and hydrolysis, and a slow exothermic phase, which is associated with product-release steps. These results are discussed in terms of their implications for energy transduction.
T a n s d u c t i o n of the chemical energy of ATP hydrolysis to mechanical energy in the actomyosin ATPase system is not understood at the molecular level. Several years ago it was suggested that the transfer of free energy between ATP and myosin occurred in the binding process (Morales, 1975; Morales & Botts, 1956). From considerations of the overall standard free energy it was possible to determine that the standard free energy of ATP hydrolysis on the enzyme surface was small. Indeed, kinetic analysis of the actomyosin ATPase has shown that the equilibrium constant for hydrolysis on the enzyme surface is only 9 at 22 “C (Bagshaw & Trentham, 1973). Energy transduction would be possible if a portion of the free energy of this strong binding interaction, K N 10” M-’ (Wolcott & Boyer, 1974; Goody et al., 1977), were retained in the myosin structure to be released as mechanical work when the product complex engages, Le., forms a cross bridge, with actin. For most chemical processes our understanding is sharpened if we know how the free energy is partitioned between enthalpy and entropy. In the study of energy transduction in muscle this is of particular interest as these thermodynamic parameters will allow us to use our intuition to postulate new or reaffirm old transduction mechanisms. In earlier calorimetric experiments, Yamada et al. (1973) measured the enthalpy of ATP hydrolysis by heavy meromyosin and noted that heat production lagged behing hydrolysis as measured by phosphate release. These workers suggested that an energy-storing product complex was formed, although no assessment of enthalpy development at early times was made. Coupled with Morales’ earlier suggestion, this prompted us to look closely at the enthalpy of binding, the first step in the myosin ATPase mechanism. In the present study we have made measurements on the enthalpy of ADP and AMP-P(NH)P’ binding to myosin. The binding enthalpy for ADP is moderately exothermic and typical of enthalpies of inhibitors binding to enzymes. In contrast, the enthalpy of binding AMP-P(NH)P, a nonhydrolyzable ATP analogue, to myosin is endothermic. We have also followed the rate of enthalpy production for the reaction of ATP and myosin as a function of time. Heat production is resolved into a fast phase, which includes binding and hydrolysis, and a slow phase, which is associated with product release. Some implications for energy transduction are presented.
Materials and Methods
From the Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242. Receiaed December I , 1978. This work was supported by a grant from the National Science Foundation (PCM 77-08087).
0006-2960/79/0418-3654$01.OO/O
The chemicals used in all experiments were of reagent grade and used without purification unless noted. Disodium salts of ATP and ADP were obtained from Sigma Chemical Co. AMP-P(NH)P was obtained from ICN and P-L Biochemicals. The purity of these nucleotides was assessed by chromatography on PEI-cellulose plates. ATP and ADP were greater than 98% pure, and AMP-P(NH)P was greater than 90% pure. A M P deaminase was obtained from Sigma Chemical Co. All solutions were prepared by using glass-distilled water. Myosin. Rabbit skeletal myosin was prepared and characterized as described earlier (Goodno & Swenson, 1975). Myosin was stored at 4 OC as a pellet precipitated from ammonium sulfate. The purity of the myosin was assumed to be 90-95% and was routinely examined by NaDodS04polyacrylamide electrophoresis. The myosin concentration in the solutions was determined by ultraviolet (Ezsol% = 5.5) and by the biuret assay which was standardized with bovine serum albumin. The calcium and EDTA ATPase activities of myosin were assayed under the conditions of Kielley & Bradley (1956) and Kielley et al. (1956). Specific activities were 0.90 f 0.02 (SEM) and 3.04 f 0.08 (SEM) hmol of Pi min-l mg-’ for calcium and EDTA assays, respectively, for 20 preparations of myosin. Myosin solutions were routinely tested for myokinase activity by a coupled assay using A M P deaminase. Most myosin samples showed no myokinase activity but Ap,A was used in the solution to assure that no spurious heat would be developed in the myosin-ADP binding experiments. Solutions for Calorimetry. Myosin stock solutions were prepared by homogenizing a predetermined weight of myosin pellet into 20 mM Tris or Pipes buffer which contained 500 mM KCl and 10 mM MgC12 at pH 7.8. This solution was extensively dialyzed vs. this same buffer until the pH values agreed to f0.005 pH unit. The concentration of the myosin stock ranged from 5 to 9 mg/mL, which corresponds to a site concentration of 21-38 FM, assuming two sites per 470000 daltons (Godfrey & Harrington, 1970). ApSA was added to a concentration of 0.02 mM from a stock solution in the same buffer for which the pH was adjusted to match the outer dialysate. Stock nucleotide solutions were prepared in the same buffer,
’
Abbreviations used: AMP-P(NH)P, 5’-adenylyl imidodiphosphate; EDTA, ethylenediaminetetraacetic acid; Tris, 2-amino-2-(hydroxymethyl)- 1,3-propanediol; Pipes, piperazine-NJV’-bis(2-ethanesulfonicacid); ApSA, P’,Ps-di(adenosine-5’) pentaphaphate; NaDcdS04,sodium dodwyl sulfate; PEI, poly(ethy1enimine).
0 1979 American Chemical Society
ENTHALPY OF NUCLEOTIDES BINDING TO MYOSIN
VOL.
18,
NO.
17,
1979
3655
n
iw 1
402
2 088
b.
t
3 097
0495
min) Reoction Heal (pv min) =
0
10
20
3.09 = 4.02 - 0.93
30
40
50
TIME (Minuled
FIGURE1: Typical calorimetric trace for the binding of ADP to myosin. The calorimeter contained 36.8 nmol of nucleotide binding sites (myosin) and 100 nmol of ADP. The reaction was initiated at the arrow (1) by rotation of the calorimeter. Rotations 2-4 provide values for the mechanical heat. The three mechanical heats were averaged and subtracted from rotation 1 to give the reaction heat for the experiment. Heats of dilution for myosin and nucleotide (generally less than 0.2 mJ) were determined in a similar procedure and were combined with this data and the calibration factor, 0.444 mJ (wV min) to yield the enthalpy of binding.
and the pH values were adjusted to h0.005 pH units of the outer dialysate. Concentrations were calculated from the measured ultraviolet absorption of adenine (em = 15.4 X lo3 cm-' M-l) and were 200-250 pM. Lower nucleotide concentrations were achieved by dilution of this stock solution. Ap,A was added to these solutions to a concentration of 0.02 mM. Calorimeter. The calorimeter was of the twin heat-conduction design and was constructed in our laboratory. The calorimetric vessels were made of gold and typically mixed 1.0 mL (enzyme) and 0.5 mL (nucleotide) of reactant solutions. Measured heats ranged from 0 to f1200 mJ. Calibration of the calorimeter was accomplished by using the neutralization of Tris by hydrochloric acid (AHi = 48.13 kJ mol-' at 15 "C). The calorimeter inherently measures the rate of heat production, and thus the quantity of heat is proportional to the integral of the voltage-time trace. In operation, the voltage output from the calorimeter was fed to an interface which performed the analogue to digital conversion, and the digitized output was stored in a Monroe 1860 programmable calculator. When data collection was complete, the computer performed the integration and output the heat equivalent in microvolt minutes. This quantity multiplied by the calibration factor gave the measured heat in millijoules. In a typical experiment the reactant solutions were loaded into the two compartments of the calorimeter cell. After allowing 1-2 h for temperature equilibration, which is indicated by a "flat" base line (
